The long-term goal of this work is to understand the retina as a neuronal machine, much like the rest of the brain for which it serves as a model. Synaptic transmission between amacrine cells forms the focus of this proposal since there is evidence that novel mechanisms of transmitter release are present in these cells. Elucidation of these fundamental mechanisms will help in understanding the design and role of this class of neuron in the retina and will likely have wide relevance to synaptic transmission throughout the brain. The experiments proposed here will combine patch clamp electrophysiology with high resolution calcium imaging of isolated, cultured amacrine cells to achieve 3 specific aims. Our first aim is to determine the relationship between calcium release from internal stores and synaptic transmission. At present the connection between calcium release and transmitter release is strong but essentially circumstantial. We will establish a causal connection by pharmacologically interfering with calcium release and showing that this alters synaptic transmission. Using light and electron microscopy we will examine the spatial relationship between calcium stores and synapses to see if it is consistent with a functional relationship. Our second aim is to define the mechanism of the calcium amplifier in dendrites that takes the small amount of calcium admitted through calcium channels and boosts it with calcium from internal stores. To achieve this aim we will use calcium imaging to examine, through the use of selective blockers, the interaction between Ryanodine receptors and IP3 receptors, both of which we know are involved in the calcium amplifier. We will examine the biochemical pathway producing IP3, again using pharmacological agents. The storage capacity of the calcium stores and the relative independence of adjacent stores will be addressed. Our third aim is to understand the role of calcium entry through TRP channels for the release of transmitter. We have preliminary evidence that these calcium permeable but voltage independent channels admit calcium to the dendritic cytoplasm when internal stores are depleted, and perhaps under other circumstances too. To reveal their role in transmitter release we will block calcium entry through these channels while monitoring synaptic transmission. [unreadable] [unreadable]